Obesity is a serious public health issue in the United States 1 and worldwide 2 and is a major risk factor for hypertension, arrhythmia, myocardial ischemia, heart failure1, 3-6, and diabetes4, 7. In obesity, parasympathetic nervous system (PSNS) regulation of the heart is markedly attenuated 8-10, which may contribute to obesity-related arrhythmia and myocardial ischemia8, 11-13. To date, however, the mechanisms of the reduced PSNS function on the heart in obesity are still poorly understood. Acetylcholine (Ach) is a major neurotransmitter of PSNS. Vesicular acetylcholine transporters (VAChT) are responsible for Ach packaging In PSNS cholinergic terminals. High plasma cholesterol (hypercholesterolemia) affects neurons in the brain14-16 but its effect on the peripheral cholinergic system is unknown. Our recent study identified mono-ubiquitination is involved in VAChT recycling and Ach uptake activity. In high fat diet (HFD)-induced obese mice, VAChT ubiquitination is reduced, along with the attenuated PSNS regulation of the heart. This study is to test a central hypothesis that Attenuated PSNS action on the heart in obesity is related to the inhibitory effect of high cholesterol on the ubiquitination-mediated VAChT recycling, leading to the reduced VAChT level and function in synaptic vesicles and decreased Ach release in PSNS cholinergic terminals. Aim 1 will test whether the attenuated PSNS effect on the heart in HFD-induced obese mice involves reduced Ach release and VAChT expression at parasympathetic terminals in the heart. Mice fed a regular diet (RD) or HFD for 6 weeks will be used as an obesity model. Vagal stimulation (VS)-induced bradycardia and Ach release in atria, and VAChT protein levels and Ach uptake activity in synaptic vesicles from PSNS cardiac terminals will be examined. Aim 2 will elucidate whether high cholesterol plays a role in the PSNS cholinergic dysfunction in the heart. Mice fed RD, RD plus Statin, HFD, HFD plus Statin, RD plus 2% cholesterol and RD plus 2% cholesterol plus Statin will be used. Responses to VS and VAChT level and Ach uptake in the PSNS cardiac terminals will be examined and correlated with cholesterol levels. The direct effect of cholesterol on VAChT expression and Ach uptake will also be tested in a cholinergic cell line, SN56. Aim 3 will delineate whether high cholesterol inhibits the mono-ubiquitination-mediated VAChT recycling and thus function in the PSNS terminals. Correlation of plasma cholesterol levels with mono- ubiquitination and Ach uptake activity of VAChT will be analyzed in atria samples from the six groups in Aim 2; The direct inhibitory effect of cholesterol on VAChT mono-ubiquitination, recycling and activity will be tested using SN56 cells. Overall, results of this study will demonstrate that the negative effect of high cholesterol on the ubiquitination-mediated VAChT recycling contributes to the reduced PSNS cholinergic action on the heart in obesity. This novel concept may open a new area of research and may suggest novel strategies for prevention and treatments of obesity-related autonomic and cardiovascular disorders.